Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae)

Supercritical CO2 extraction of Eucalyptus leaves oil and comparison with Soxhlet extraction and hydro-distillation methods

The urinary metabolites of the monoterpene, p-cymene, were studied in three marsupial species: a generalist herbivore, the brushtail possum (Trichosurus vulpecula), and two specialist folivores, the greater glider (Petauroides volans) and the ringtail possum (Pseudocheirus peregrinus), as well as in the laboratory rat (Rattus norvegicus). Each animal was dosed, intragastrically, with single doses of p-cymene (0.37 mmol/kg and/or 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods. Quantitative analysis of urinary metabolites by extraction, gas chromatography, and mass spectrometry gave a mean recovery of 64% (range 52–74%) of the administered dose in 48 hr for the four species. No fecal metabolites were found. A species-specific pattern of metabolite excretion was evident and reflected the natural occurrence of p-cymene (and terpenes in general) in the diet. If the metabolites excreted are grouped according to the total number of oxygen atoms added (one to four), then the rat excreted metabolites encompassing all degrees of oxidation, but predominantly a monooxygenated metabolite. The brushtail possum excreted metabolites having two, three, and four oxygen atoms added. The ringtail possum and greater glider only excreted metabolites with three or four oxygen atoms. Conjugation played a significant role in the excretion of oxidized metabolites in only the brushtail possums and the rat. These findings indicate that species encountering terpenes, such as p-cymene, in their normal diet have developed efficient oxidation pathways to eliminate them. This oxidative efficiency could also reduce the necessity for subsequent conjugation of metabolites which minimizes further demands on a nutritionally limited diet.

Comparative Metabolism of Dietary Terpene, p-Cymene, in Generalist and Specialist Folivorous Marsupials

Intraspecific variation in leaf oils of Melaleuca alternifolia (Myrtaceae)

Plant terpenes play many roles in natural systems, from altering plant–animal interactions, to altering the local abiotic environment. Additionally, many industries depend on terpenes. For example, commercially used essential oils, including tea tree oil and lavender oil, are a mixture of terpenes. Many species of the family Myrtaceae form a key resource for these industries due to the high concentration of terpenes found predominately in their leaves. The frequency of chemotypic differences within many species and populations can lead to costly errors in industry. Terpene diversity in Myrtaceae is driven by variation in the terpene synthase enzymes, which catalyse the conversion a few common substrates into thousands of terpene structures. We review terpene diversity within and between species of Myrtaceae and relate this to variation in the terpene synthase enzymes to reconstruct the evolution of foliar terpene diversity in Myrtaceae. We found that (1) high inter- and intra-species variation exists in terpene profile and that α-pinene the most likely ancestral foliar terpene, and (2) that high concentration of 1,8-cineole (a compound which is regarded as the signature compound of Myrtaceae) is limited to just four Myrtaceae sub-families. We suggest that the terpene synthase enzymes do not limit terpene diversity in this family and variation in these enzymes suggests a mode of enzymatic evolution that could lead to high 1,8-cineole production. Our analysis highlights the need to standardise methods for collecting and reporting foliar terpene data, and we discuss some methods and issues here. Although there are many gaps in the published data, our large scale analysis using the results of many studies, shows the value of a family wide analysis for understanding both the evolution and industrial potential of terpene-producing plants.

The evolution of foliar terpene diversity in Myrtaceae

The vapour-phase chromatographic analysis of the ethanol extract of leaves of Eucalyptus species has been investigated in order to develop a more accurate determination of the terpene content. Conventional techniques based on steam distillation have been shown to have serious faults when compared with solvent extraction procedures. The convenience of the method during both the field sampling and the laboratory analysis stages is a major advantage. The procedure is suitable for large numbers of samples and requires only small amounts of leaf material.

Rapid and accurate determination of terpenes in the leaves of Eucalyptus species

A simple and rapid method has been developed to determine the water content of leaves of Eucalyptus species. The procedure described should be applicable without significant changes to most other species, and is particularly valuable for all those which lose volatile oils during drying. The method, which is based on solvent extraction followed by Karl Fischer titration, has proved successful for several species, experimental results confirming that no components of the leaf extract significantly interfere with the water determination. The simplicity and convenience of the solvent extraction method has been shown to have definite advantages over conventional procedures such as loss of mass on oven drying.

Donald G. Ammon

Papers

Rapid and accurate determination of terpenes in the leaves of Eucalyptus species

Rapid and accurate chemical determination of the water content of plants containing volatile oils